Use of isolated domains of type IV collagen to modify cell and tissue interactions

ABSTRACT

The instant invention provides methods and kits for inhibiting angiogenesis, tumor growth and metastasis, and endothelial cell interactions with the extracellular matrix, involving contacting the tumor or animal tissue with at least one isolated type IV collagen NC1 α chain monomer. In a specific embodiment of the invention, the isolated domain of type IV collagen comprises the NC1 (α1), (α2), (α3), or (α6) chain monomer, or protein constructs having substantially the same structure as the NC1 (α1), (α2), (α3), or (α6) chain monomer.

CROSS REFERENCE

The present application is a continuation of Ser. No. 09/277,665 filedMar. 26, 1999, which is a continuation in part of U.S. patentapplications Ser. No. 60/106,170 filed Oct. 29, 1998; Ser. No.60/079,783 filed Mar. 27, 1998; and Ser. No. 09/183,548 filed Oct. 30,1998, now abandoned which is a continuation of U.S. application Ser. No.08/800,965 filed Feb. 18, 1997, now U.S. Pat. No. 5,856,184, which is acontinuation of U.S. application Ser. No. 08/497,206 filed Jun. 30, 1995now U.S. Pat. No. 5,691,182, all of which are herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

This invention relates to methods and kits for inhibiting angiogenesis,tumor growth and metastasis, and endothelial cell interactions with theextracellular matrix.

BACKGROUND OF THE INVENTION

Angiogenesis, the process of formation of new blood vessels, plays animportant role in physiological processes such as embryonic andpostnatal development as well as in wound repair. Formation of bloodvessels can also be induced by pathological processes involvinginflammation (e.g., diabetic retinopathy and arthritis) or neoplasia(e.g., cancer); (Folkman, 1985, Perspect, Biol. Med., 29, 10).Neovascularization is regulated by angiogenic growth factors secreted bytumor or normal cells as well as the composition of the extracellularmatrix and by the activity of endothelial enzymes (Nicosia andOttinetti, 1990, Lab. Invest., 63, 115).

During the initial stages of angiogenesis, endothelial cell sproutsappear through gaps in the basement membrane of pre-existing bloodvessels (Nicosia and Ottinetti, 1990, supra; Schoefl, 1963, VirehousArch, Pathol. Anat. 337, 97-141; Ausprunk and Folkman, 1977, Microvasc.Res. 14, 53-65; Paku and Paweletz, 1991, Lab. Invest. 63, 334-346). Asnew vessels form, their basement membrane undergoes complex structuraland compositional changes that are believed to affect the angiogenicresponse (Nicosia, et. al., 1994, Exp. Biology, 164, 197-206). Earlyplanar culture models have shown that basement membrane moleculesmodulate the attachment, migration and proliferation and organizationalbehavior of endothelial cells (Nicosia, et. al., 1994, supra). Morerecent studies with three-dimensional aortic culture models that moreclosely simulate angiogenic conditions during wound healing in vivosuggest that basement membrane is a dynamic regulator of angiogenesiswhose function varies according to its molecular components (Nicosia,1994, supra).

A common feature of all solid tumor growth is the requirement for ablood supply. Therefore, numerous laboratories have focused ondeveloping anti-angiogenic compounds based on growth factors and theirreceptors. While this approach has led to some success, the number ofgrowth factors known to play a role an angiogenesis. is large.Therefore, the possibility exists that growth factor antagonists mayhave only limited use in treating cancer since tumors and associatedinflammatory cells likely produce a wide variety of factors that caninduce angiogenesis.

In this regard, a strategy that targets a common feature ofangiogenesis, such as endothelial cell adhesion to the extracellularmatrix (ECM), might be expected to have a profound physiological impacton tumor growth in humans. This notion is supported by the fact thatantagonists of specific ECM cell adhesion receptors such as αvβ3 andαvβ5 integrins can block angiogenesis. Furthermore, the αvβ3 integrin isexpressed most prominently on cytokine—activated endothelial and smoothmuscle cells. and has been shown to be required for angiogenesis.(Varner et al., Cell Adhesion and Communication 3:367-374 (1995); Brookset al., Science 264:569-571 (1994)). Based on these findings, apotentially powerful new approach to anti-angiogenic therapy might be tospecifically target critical regulatory domains within distinct ECMcomponents.

The basement membrane (basal lamina) is a sheet-like extracellularmatrix (ECM), which is a basic component of all tissues. The basallamina provides for the compartmentalization of tissues, and acts as afilter for substances traveling between tissue compartments. Typicallythe basal lamina is found closely associated with an epithelium orendothelium in all tissues of an animal including blood vessels andcapillaries. The basal lamina components are secreted by cells and thenself assemble to form an intricate extra-cellular network. The formationof biologically active basal lamina is important to the development anddifferentiation of the associated cells.

Type IV collagen has been shown to be a major structural component ofbasement membranes. The protomeric form of type IV collagen is formed asa heterotrimer made up from a number of different subunit chains calledα1(IV) through α6(IV). Up to now, six genetically distinct α-chainsbelonging to two classes with extensive homology have been identified,and their relative abundance has been demonstrated to be tissuespecific. The type IV collagen heterotrimer is characterized by threedistinct structural domains: the non-collagenous (NC1) domain at thecarboxyl terminus; the triple helical collagenous domain in the middleregion; and the 7S collagenous domain at the amino terminus. (Martin,et. al., 1988, Adv. Protein Chem. 39:1-50; Gunwar, et. al. 1991, J.Biol. Chem. 266:14088-14094).

The capability of expression of recombinant α(IV) NC1 domains providesthe opportunity to study the effect of specific domains on manybiological processes, such as angiogenesis, tumor metastasis, cellbinding to basement membranes, and assembly of Type IV collagenmolecules.

SUMMARY OF THE INVENTION

The instant invention provides methods and kits for inhibitingangiogenesis, tumor growth and metastasis, and endothelial cellinteraction with the extracellular matrix, each method comprisingcontacting the tumor or animal tissue with an one or more isolated typeIV collagen NC1 α chain monomer selected from the group consisting ofα1, α2, α3, and α6 NC1 chain monomers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effects of NC1 (Hexamer) and 7S domains of TypeIV collagen at a 50 μg/ml concentration on angiogenesis from mousethoracic aorta organ cultures.

FIG. 2 illustrates the effects of 7S domain of Type IV collagen onangiogenesis from mouse thoracic aorta organ cultures. The domainconcentrations employed in this experiment were 0 μg/ml (control); 0.5μg/ml; 5 μg/ml and 50 μg/ml.

FIG. 3 illustrates the effects of NC1 (Hexamer) domain of Type IVcollagen on angiogenesis from mouse thoracic aorta organ cultures. Thedomain concentrations employed in this experiment were 0 μg/ml(control); 5 μg/ml and 5 μg/ml and 50 μg/ml.

FIG. 4 are photographs of mouse thoracic aorta segments embedded inMatrigel (EHS basement membrane matrix, Collaborative BiomedicalProducts, Bedford, Mass.) at 5 days of culture. Control specimen (0μg/ml of NC1 (Hexamer) and 7S domains) exhibited growth of microvesselsfrom the cultured tissue into the matrix (FIG. 4A). In contrast,angiogenesis was inhibited in specimens cultured with 50 μg/ml of 7Sdomain (FIG. 4B) and NC1 (Hexamer) domain (FIG. 4C).

FIG. 5 is a graphical representation of data demonstrating the in vivoeffect of IV injection of recombinant (α1) type IV collagen monomer onangiogenesis using fibrin implants in rats.

FIG. 6 is a graphical representation of data demonstrating that therecombinant (α1) and (α2) NC1 monomers inhibit the bFGF-induced increasein angiogenic index in vivo.

FIG. 7 is a graphical representation of demonstrating the dose responseeffect of recombinant (α2) NC1 monomer on the bFGF-induced increase intotal blood vessel branch points in vivo.

FIG. 8 is a graphical representation of data demonstrating the doseresponse effect of recombinant (α2) NC1 monomer on the bFGF-inducedincrease in angiogenic index in vivo.

FIG. 9 is a graphical representation of data demonstrating the doseresponse effect of recombinant (α2) NC1 monomer on the bFGF-inducedincrease in angiogenic index in vivo.

FIG. 10 is a graphical representation of data demonstrating the effectof recombinant (α1) and (α2) NC1 monomers on mean CS-1 melanoma tumorweight in vivo.

FIG. 11 is a graphical representation of data demonstrating the doseresponse effect of recombinant (α2) NC1 monomer on mean CS-1 melanomatumor weight in vivo.

FIG. 12 is a graphical representation of data demonstrating the effectof recombinant (α1), (α2), and (α4) NC1 monomers on mean HT1080 tumorweight in vivo.

FIG. 13 is a graphical representation of data demonstrating the effectof recombinant (α1), (α2), (α3) and (α5) NC1 monomers on mean HEP-3tumor weight in vivo.

FIG. 14 is a graphical representation of data demonstrating humanendothelial cell adhesion to immobilized NC1 α monomers.

FIG. 15 is a graphical representation of data demonstrating the effectof soluble α1 and α2 NC1 monomers on human endothelial cell adhesion topepsinized collagen type IV.

FIG. 16 is a graphical representation of data demonstrating the effectof isolated recombinant NC1 monomers on human endothelial cell migrationin vitro.

FIGS. 17 A-F provides the sequences of each type IV collagen a chainmonomer.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.).

As used herein, the term Type IV collagen domain encompasses the groupof molecules including the non-collagenous NC1 domain (Hexamer) and 7Scollagenous domains, as well as NC1 α chain monomers.

The invention comprises methods for using Type IV collagen NC1α-monomers (ie: α1, α2, α3, and α6), which are defined to include suchmonomers isolated from any multicellular organism or produced viarecombinant protein expression from a gene encoding such a monomer fromany multicellular organism, and also to encompass various modifications,additions, and/or deletions to such monomers.

In one aspect, the present invention provides methods and kits forinhibiting angiogenesis in an animal tissue comprising contacting thetumor or animal tissue with an amount effective to inhibit angiogenesisof a polypeptide composition comprising one or more isolated type IVcollagen NC1 α chain monomer selected from the group consisting of α1,α2, α3, and α6 NC1 chain monomers.

In another aspect, the present invention provides methods and kits forinhibiting tumor growth in tissue comprising contacting the tumor ortissue with an amount effective to inhibit tumor growth of a polypeptidecomposition comprising one or more isolated type IV collagen NC1 α chainmonomer selected from the group consisting of α1, α2, α3, and α6 NC1chain monomers.

In another aspect, the present invention provides methods and kits forinhibiting tumor metastasis in tissue comprising contacting the tumor ortissue with an amount effective to inhibit metastasis of a polypeptidecomposition comprising one or more isolated type IV collagen NC1 α chainmonomer selected from the group consisting of α1, α2, α3, and α6 NC1chain monomers.

In a further aspect, the present invention provides methods and kits forinhibiting endothelial cell interactions with the extracellular matrixin tissue comprising contacting the tumor or tissue with an amounteffective to inhibit endothelial cell interactions with theextracellular matrix of a polypeptide composition comprising one or moreisolated type IV collagen NC1 α chain monomer selected from the groupconsisting of α1, α2, α3, and α6 NC1 chain monomers.

The NC1-encoding domain of each of the six a chain cDNAs has been clonedinto a vector for recombinant protein expression as previously described(Sado et al., Kidney Intl. 53:664-671 (1998), incorporated by referenceherein in its entirety). The vectors are used to stably transfect humankidney 293 cells, which produce the recombinant protein. The DNA anddeduced amino acid sequences of the recombinant type IV collagen alphachain monomers produced as described are shown in FIGS. 17A-F. The first17 amino acids corresponds to a BM40 signal sequence (which is cleavedfrom the mature protein), to facilitate protein secretion. All thesecreted proteins (ie: mature proteins) start with the sequence APLAfollowed by the affinity tag, DYKDDDDK at the amino terminus. This tagfacilitates purification and identification of the material, and doesnot interfere with biological activity of the recombinant NC1 α chainmonomer.

The type IV collagen NC1 α chain monomers can be produced by any methodknown in the art, including using recombinant DNA technology orbiochemical peptide synthesis technology, or by isolating the NC1domains from animal sources, such as from basement membrane sources suchas bovine lens capsule and bovine kidney glomeruli. (Peczon et al., Exp.Eye Res. 30:155-165 (1980); Langeveld et al., J. Biol. Chem.263:10481-10488 (1988); Gunwar et al., J. Biol. Chem. 266:14088-14094(1991))

In practicing the invention, the amount or dosage range of type IVcollagen NC1 α chain monomers employed is one that effectively inhibitsangiogenesis, tumor growth, tumor metastasis, and/or endothelialcell-extracellular matrix interactions. An inhibiting amount of NC1 αchain monomers that can be employed ranges generally between about 0.01μg/lkg body weight and about 10 mg/kg body weight, preferably rangingbetween about 0.05 μg/kg and about 5 mg/kg body weight.

The NC1 α chain monomers may be administered by any suitable route,including orally, parentally, by inhalation spray, rectally, ortopically in dosage unit formulations containing conventionalpharmaceutically acceptable carriers, adjuvants, and vehicles. The termparenteral as used herein includes, subcutaneous, intravenous,intraarterial, intramuscular, intrastemal, intratendinous, intraspinal,intracranial, intrathoracic, infusion techniques or intraperitoneally.In preferred embodiments, the NC1 α chain monomers are administeredintravenously or subcutaneously.

The NC1 α chain monomers may be made up in a solid form (includinggranules, powders or suppositories) or in a liquid form (e.g.,solutions, suspensions, or emulsions). The NC1 α chain monomers of theinvention may be applied in a variety of solutions. Suitable solutionsfor use in accordance with the invention are sterile, dissolvesufficient amounts of the NC1 α chain monomers, and are not harmful forthe proposed application.

The NC1 α chain monomers may be subjected to conventional pharmaceuticaloperations such as sterilization and/or may contain conventionaladjuvants, such as preservatives, stabilizers, wetting agents,emulsifiers, buffers etc.

For administration, the NC1 α chain monomers are ordinarily combinedwith one or more adjuvants appropriate for the indicated route ofadministration. The compounds may be admixed with lactose, sucrose,starch powder, cellulose esters of alkanoic acids, stearic acid, talc,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulphuric acids, acacia, gelatin, sodium alginate,polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted orencapsulated for conventional administration. Alternatively, thecompounds of this invention may be dissolved in saline, water,polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidalsolutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil,tragacanth gum, and/or various buffers. Other adjuvants and modes ofadministration are well known in the pharmaceutical art. The carrier ordiluent may include time delay material, such as glyceryl monostearateor glyceryl distearate alone or with a wax, or other materials wellknown in the art.

The present invention may be better understood with reference to theaccompanying examples that are intended for purposes of illustrationonly and should not be construed to limit the scope of the invention, asdefined by the claims appended hereto.

EXAMPLE 1 In Vitro Effect on Angiogenesis

With modifications, the procedures of Nicosia and Ottinetti (1990),supra, and Nicosia, et. al. (1994), supra, were utilized for experimentsdesigned to test the effect of Type IV collagen on angiogenesis under invitro conditions. The model has been used to study the effects of growthfactors and extracellular matrix molecules on the angiogenic responseand employs aortic rings cultures in three-dimensional collagen gelsunder serum-free conditions. These experiments are outlined below.

A. Methods

Experiments were performed with 1-3 month old Swiss Webster male mice.Following anesthesia, the thoracic aorta was excised under asepticconditions and transferred to sterile MCDB 131 sterile growth medium(Clonetics, San Diego, Calif.) containing antibiotics. Fat was dissectedaway from the aorta and approximately six to eight 1 mm thoracicsegments were obtained from each specimen. Segments were transferred to48 well tissue culture plates. The wells of these plates were layeredwith 100 microliters of Matrigel (EHS basement membrane, CollaborativeBiomedical Products, Bedford, Mass.) prior to transfer of the aorticsegments. The Matrigel was diluted 1:1 with MCDB 131 growth medium priorto use. The segments were centered in the wells and an additional 100microliters of Matrigel was then placed over the specimens. The aorticsegments were therefore embedded in the basement membrane matrix. Eachwell then received 300 microliters of MCDB 131 growth medium. The plateswere placed in an incubator maintained at 37° C. with 5% CO₂. Specimenswere observed daily over a 7 day period. Newly growing microvessels werecounted using an inverted phase microscope at various times during theculture period, but data is expressed at 3 and 5 days of culture. Totest for the effect of Type IV collagen on angiogenesis, domains atknown concentrations were mixed with the Matrigel and with the MCDB 131growth medium. Fresh MCDB 131 growth medium (plus and minus collagendomains) was changed every 3 days.

B. Results

After establishing the time course of angiogenesis under controlconditions (Matrigel plus MCDB 131 growth medium), experiments wereperformed using various concentrations of Type IV collagen (isolatedfrom bovine lens) NC1 (hexamer) and 7S domains. Data represents theanalysis of at least 3 specimens per experimental condition. In thefirst experiment (FIG. 1), analysis indicated that at a concentration of50 μg/ml, NC1 domain and 7S domain significantly inhibited angiogenesisas monitored at 3 and 5 days of culture. In the second experiment,various concentrations of these domains were analyzed. As indicated inFIG. 3, 7S domain at 50 μg/ml again significantly inhibited angiogenesisat 3 and 5 days. Inhibition was reduced at 5 and 0.5 μg/mlconcentrations. As indicated in FIG. 2, NC1 domain was less effective inblocking angiogenesis as compared to that observed in the firstexperiment (FIG. 1), although it was still effective. In addition, ascompared to the 7S domain, there was less of a correlation betweenconcentration and inhibitory action.

FIGS. 4A-C are photographs of mouse thoracic aorta segments embedded inMatrigel (EHS basement membrane matrix, Collaborative BiomedicalProducts, Bedford, Mass.) at 5 days of culture in the presence orabsence of 50 μg/ml of Type IV collagen domains. The control specimen(no domains) exhibited growth of microvessels from the cultured tissueinto the matrix (FIG. 4A). In contrast, angiogenesis inhibition wasobserved in tissues cultured in the presence of 50 μg/ml of 7S (FIG. 4B)and NC1 (Hexamer) domain (FIG. 4C).

EXAMPLE 2 Subcutaneous Fibrin Implant Angiogenesis

Recombinant human type IV collagen NC1 (α3) monomer (Sado et al., KidneyInternational 53:664-671 (1998)) was injected intravenously in Fisher344 rats containing fibrin implants surgically placed subcutaneously, amodified version of the method described by Dvorak et al (Lab. Invest.57(6):673-686 (1987)). The implants were then removed and directlyanalyzed using an inverted microscope. The analysis involved countingthe number of blood vessels that had grown into the fibrin in thecontrol and experimental group.

Briefly, 4 fibrin implants were surgically implanted subcutaneously intoFisher 344 rats (2 dorsal and 2 ventral sides). The average rat weightwas approximately 125 grams.

Three rats (EXP) were given tail vein injections of either control(fibrin alone), 100 μl of 100 μg/ml of 7S domain of type IV collagen(approximately 0.80 mg/kg body weight), 100 μl of 100 μg/ml of type IVcollagen hexamer (approximately 0.80 mg/kg body weight), or recombinantcollagen type IV NC1 (α3) monomer at a concentration of 1.26 mg/ml inPBS (120 g protein, or approximately 0.96 mg/kg body weight) and 3 rats(C) were given 100 μl tail vein injections of PBS. Injections ofrecombinant protein were given every other day for five doses. Theinjection schedule was as follows:

Day 1: (implant day) injection and remove blood sample (EXP and C)

Day 3: Injection (EXP and C)

Day 5: Injection and remove blood sample (EXP and C)

Day 7: Injection (EXP and C)

Day 9: Injection and remove blood sample (EXP and C)

Day 11: Remove and fix implants (save blood sample) (EXP and C)

The results of one experiment were as follows:

2 week in vivo experiment: Control (fibrin alone) about 66 BV 7S domainof type IV lens collagen (100 μg/ml) None Hexamer of type IV lenscollagen (100 μg/ml) None Monomer (α3) None

The results are shown as the mean number of blood vessels per implant.The results of this study demonstrate that isolated domains of type IVcollagen, including the α3 monomer, can significantly inhibit capillarygrowth in the in vivo fibrin clot implant model. In subsequentexperiments, the inhibitory effect was occasionally, seen to attenuatewith time, suggesting that higher dosages or more frequent injectionsmight be even more effective.

A similar experiment was conducted using recombinant human type IVcollagen NC1 (α1) monomer (100 μl of a 1 μg/μ1 solution; approximately0.80 mg/kg body weight) and comparing the number of blood vessels thathad grown into the fibrin at day 11 of treatment relative to the controlgroup. Three rats per group were analyzed with each rat having 4implants. These experiments demonstrated that administration of the almonomer significantly inhibited capillary growth in the in vivo fibrinclot implant model (FIG. 5).

EXAMPLE 3 Recombinant NC1 (α2) Domain Inhibits Angiogenesis in vivo

We next tested the effects of systemic administration of soluble NC1α-chain monomers in the chick embryo CAM angiogenesis assay.

Angiogenesis was induced in the CAMs of 10 day old chick embryos withbFGF as described (Brooks et al., Cell 92:391-400 (1998)). Twenty fourhours later the embryos were systemically treated with variousconcentrations of recombinant NC1 (α-chain monomers, in a total volumeof 100 μl of sterile phosphate buffered saline (PBS). Two days later theembryos were sacrificed and the filter discs and CAM tissues removed.Angiogenesis was quantitated by counting the number of angigogenic bloodvessel branch points in the confined area of the filter disc. TheAngiogenic Index is defined as the number of branch points fromexperimental treatment minus control treatment.

In initial experiments, recombinant α1 or α2 NC1 domains were injectedat a concentration of 50 μg per embryo. At this concentration, the NC1domains were shown to be highly toxic as demonstrated by greater than90% embryo cell death. However, at lower doses they were well toleratedand showed potent anti-angiogenic activity. A total of 6 individualangiogenesis experiments were conducted with the NC1 domains. However,in two experiments, the bFGF induction was low, making it difficult tointerpret the results. The NC1 α2 domain appeared to be more consistentand potent than the α1 NC1 domain at inhibiting angiogenesis. In fact,systemic administration of 30 μg of NC1 α2 consistently inhibitedangiogenesis by greater than 90% (FIGS. 6-9), as measured by inhibitionof the bFGF-induced increase in the angiogenic index and the mean numberof blood vessel branch points. In contrast, NC1 α1 domain showedvariable inhibitory activity (0%-50%) throughout the experiments.

EXAMPLE 4 Recombinant NC1 Domain Inhibits Melanoma Tumor Growth in vivo

Since the growth of all solid tumors depends on angiogenesis to providenutrients for its continued expansion, reagents that have the capacityto inhibit angiogenesis may significantly inhibit tumor growth.Therefore, we tested the effects of recombinant NC1 domains of type IVcollagen for their effects on tumor growth in vivo.

To test the effects of NC1 domains on tumor growth in vivo, we utilizedthe chick embryo tumor growth assay. Briefly, single cell suspensions of3 distinct tumor types were applied to the CAM of 10 day old chickembryos. The tumors included CS-1 Melanoma cells (5×10⁶), HT1080 humanfibrosarcoma cells (4×10⁵) and Hep-3 human epidermoid carcinoma cells(2×10⁵). The embryos were injected systemically with varyingconcentrations of NC1 α-chain monomers 24 hours later. The embryos werenext allowed to incubate for a total of 7 days, at which time they weresacrificed. The resulting tumors were resected and wet weightsdetermined. A total of 6 tumor growth assays were conducted with the 3distinct tumor types. A single injection of 10 μg NC1 α2 domaininhibited CS1 melanoma tumor growth by approximately 70% relative tocontrol (FIG. 10). In similar experiments, dose response curves werecompleted with CS-1 tumors. Systemic administration of NC1 α2 resultedin a dose-dependent inhibition of CS-1 melanoma tumor growth in vivowith a maximum inhibition following a single dose at 30 μg (FIG. 11).Systemic administration of NC1 α1 also inhibited CS-1 tumor growth butit was variable and in some experiments failed to inhibit tumor growth(See FIG. 10). In similar experiments, NC1 α2 inhibited HT1080 humanfibrosarcoma tumor growth by approximately 50% after a single systemicinjection of 30 μg, while NC1 α1 and α4 had no effect (FIG. 12).Finally, systemic administration of NC1 α2 (30.0 μg) and α3 inhibitedHep-3 human epidermoid carcinoma tumor growth by approximately 40% and60% respectively, and α1 inhibited Hep-3 tumor growth by approximately30%, while NC1 α5 domain failed to inhibit tumor growth (FIG. 13).

We conclude from these in vivo studies that tumor growth can beinhibited by isolated NC1 α-chain monomers. These molecules can thus beused alone, or to complement the use of existing anti-tumor agents, inproviding enhanced and more effective anti-tumor therapy.

EXAMPLE 5 Immobilized NC1 Domains Support Human Endothelial CellAdhesion

In order for new blood vessels to form, endothelial cells must have thecapacity to adhere and migrate through the ECM. Moreover, thisendothelial cell-ECM interaction may facilitate signal transductionevents required for-new blood vessel formation. Therefore, since typeIV-collagen is an ECM protein which is known to support cell adhesion,we tested the ability of the NC1 domains to support endothelial cellattachment.

Microtiter plates were coated with 25 μg/ml of purified NC1 domainsfollowed by incubation with 1% bovine serum albumin (BSA) to blocknon-specific interactions. Human endothelial cells (ECV304) were thenallowed to attach to the immobilized NC1 domains for 1 hour.Non-adherent cells were removed by washing and attached cells werequantified by measuring the optical density (O.D.) of crystal violeteluted from attached cells. Data bars represent the mean +/− standarderror of the O.D. from triplicate wells.

Immobilized NC1 α2, α3, and α6 domains supported endothelial celladhesion while NC1 α1, α4, and α5 domains promoted little if any celladhesion (FIG. 14). Soluble NC1 α1 (a1) and α2 (a2) inhibitedendothelial cell adhesion to pepsinized collagen type IV byapproximately 50% (FIG. 15).

Taken together, these findings demonstrate that isolated, recombinantNC1 domains from the α1, α2, α3, and α6 chains of collagen type IV canmediate human endothelial cell adhesion and/or inhibit endothelial celladhesion to ECM proteins in vitro, and suggest that the potentanti-angiogenic and anti-tumor activity of the isolated NC1 domains isdue to disruption of endothelial cell interaction with the extracellularmatrix that are necessary for angiogenesis.

EXAMPLE 6 Endothelial Cell Migration

Invasive cellular processes such as angiogenesis and tumor metastasisalso require cellular motility. Thus we evaluated the ability ofisolated NC1 domains to support human endothelial cell migration invitro. These experiments were conducted essentially according to themethods in Brooks et al., J. Clin. Invest. 99:1390-1398 (1997).

The results of these experiments indicate that NC1 α2, α3, and α6domains can support human endothelial cell migration in vitro, while α1,α4, and α5 domains showed little if any capacity to support endothelialcell migration (FIG. 16).

EXAMPLE 7 Efficacy in Lewis Lung in vivo Tumor

The above studies indicated that specific domains of collagen type IVcan promote cell migration in vitro. Thus, we evaluated the ability ofNC1 domains to support endothelial cell migration in vivo.

The α (IV) NC1 domain hexamer, isolated by enzymatic digestion of bovinelens capsule basement membrane by known protocols (Peczon et al., Exp.Eye Res. 30:155-165 (1980)) was tested in the metastatic Lewis lungmouse tumor model using a standard protocol which is considered to be agood model of both metastasis and angiogenesis of lung tumors. (See forexample, Teicher et al., Anticancer Res. 18:2567-2573 (1998); Guibaud etal., Anticancer Drugs 8:276-282 (1997); Anderson et al., Cancer Res.56:715-718 (1996)).

Each study consisted of an untreated control group and six treatmentgroups. There were ten animals per treatment group with 40 mice in thecontrol. In each study, all treatment was administered intravenouslyonce every 2 days for 7 doses starting one day after tumor inoculation.Dosages of a (IV) NC1 hexamer were either 100 μg/mouse or 200 μg/mouse.In the Lewis lung study, the tumor cell inoculum was 1×10⁶ viable cells.All animals were weighed twice a week throughout the study. Starting oneday after the last treatment, 5 mice were periodically sacrificed fromeach control group to measure pulmonary tumor burden. The experiment wasterminated at day 14 when the lungs of the control animals hadsufficient tumor mass to provide meaningful evaluation. At that time,the lungs of all remaining animals were excised, weighed, and the numberof tumor foci greater than 2 mm in diameter counted. The resulting datashowed that both dosages of α (IV) NC1 hexamer significantly reduced thenumber of visible lung metastases (Mann-Whitney Rank Sum Test, p<0.05),with 8 visible lung metastases in the control, vs. 5 (100 μg/mouse) and4 (200 μg/mouse), and the 100 μg/mouse dosage reduced the lung weightsfrom a median of 520 mg in controls to a median of 462 mg inexperimental, while the median lung weight of mice treated with 200μg/mouse was 620 mg.

Other in vivo studies demonstrated that tumor cell metastasis to thelung can be reduced by 50% or more using intravenous injections of theType IV collagen domains in murine B16 melanoma, human A375SM melanomaxenografts. Furthermore, injection of the NC1 hexamer also significantlyreduced the number of lung tumors in separate Lewis Lung tumor studies.

We conclude from all of the above studies that angiogenesis, tumorgrowth and metastasis, and endothelial cell adhesion to the ECM, can beinhibited by isolated, recombinant domains of type IV collagen. Thepresent invention is thus broadly applicable to a variety of uses whichinclude inhibition of angiogenesis and treatment of diseases andconditions with accompanying undesired angiogenesis, such as solid andblood-borne tumors including but not limited to melanomas, carcinomas,sarcomas, rhabdomyosarcoma, retinoblastoma., Ewing sarcoma,neuroblastoma, osteosarcoma, and leukemia.

The invention is further applicable to treating non-tumorigenic diseasesand conditions with accompanying undesired angiogenesis, including butnot limited to diabetic retinopathy, rheumatoid arthritis, retinalneovascularization, choroidal neovascularization, macular degeneration.,corneal neovascularization, retinopathy of prematurity., corneal graftrejection, neovascular glaucoma., retrolental fibroplasia, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi's sarcoma, Mooren ulcer, Terrien's marginaldegeneration, marginal keratolysis, traum, systemic lupus,polyarteritis, Wegeners sarcoidosis, scleritis, Steven's Johnsondisease, radial keratotomy, sickle cell anemia, sarcoid, pseudoxanthomaelasticum, Pagets disease, vein occlusion, artery occulsion, carotidobstructive disease, chronic uveitis, chronic vitritis, Lyme's disease,Eales disease, Bechets disease, myopia, optic pits, Stargarts disease,pars planitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, post-laser complications, abnormal proliferation offibrovascular tissue, hemangiomas, Osler-Weber-Rendu, acquired immunedeficiency syndrome, ocular neovascular disease, osteoarthritis, chronicinflammation, Crohn's disease, ulceritive colitis, psoriasis.,atherosclerosis, and pemphigoid. See U.S. Pat. No. 5,712,291)

The invention is also broadly applicable to methods for inhibiting tumorgrowth and metastasis, reduction of scar tissue formation, reduction ofcomplications due to cell adhesion in organ transplants, and theinhibition of lymphocyte adhesion and mobility.

While the fundamental novel features of the invention have been shownand described, it will be understood that various omissions,substitutions, and changes in the form and details illustrated may bemade by those skilled in the art without departing from the spirit ofthe invention. For example, various modifications, additions. and/orsubstitutions can be made to the type IV collagen a monomer chains thatwould be encompassed by the invention. It is the intention, therefore,to be limited only as indicated by the scope of the following claims:

We claim:
 1. A pharmaceutical composition consisting of: a) an amounteffective of isolated NC1 α2 chain monomers of type IV collagen toinhibit angiogenesis, tumor metastasis, or tumor growth in a host inneed thereof, or to treat an angiogenesis-mediated disease or conditionin a mammal; and b) a pharmaceutically acceptable carrier.
 2. Thepharmaceutical composition of claim 1 wherein the NC1 α2 chain domain ispresent at a concentration permitting administration to a subject inneed thereof at a dosage of at least 0.01 microgram per kilogram bodyweight of the subject.
 3. The pharmaceutical composition of claim 1wherein the NC1 α2 chain domain is present at a concentration permittingadministration to a subject in need thereof at a dosage of between 0.01microgram per kilogram body weight of the subject and 10 milligrams perkilogram body weight of the subject.
 4. The pharmaceutical compositionof claim 1 wherein the NC1 α2 chain domain is present at a concentrationpermitting administration to a subject in need thereof at a dosage ofbetween 0.05 microgram per kilogram body weight of the subject and 5milligrams per kilogram body weight of the subject.